mmg_233_2013_genetics_genomicswikiaorg-20200214-history
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Lentiviral vectors (LV) have been widely used in biological research to introduce genetic material into cells. The process of introduction of foreign nucleic acids into mammalian cells is called transduction. Viral vectors, in general, have been a method of choice when nuclei acids are to be introduced in difficult-to-transfect cells. The major advantage of using LVs is their ability to transduce non-diving cells, making them one of the most efficient transducing systems. Background Lentiviruses are single stranded (ss) RNA viruses characterized by long incubation periods, i.e., post-infection with virus, it takes a long time for the symptoms to appear. All viruses under the Lentivirus genus belong to the Retroviridae family of viruses; with ‘retro’ referring to their ability to reverse transcribe their ssRNA genomes to double stranded DNA that is integrated into the host genome. Retroviruses are enveloped viruses. Lentiviruses include primate viruses such as human immunodeficiency virus (HIV-1), simian immunodeficiency virus (SIV), as well as non-primate viruses like equine infectious anemia virus, caprine arthritis-encephalitis virus, maedi-visna virus, feline immunodeficiency virus (FIV) and bovine immunodeficiency virus. All of these can be used to generate LV’s 1. HIV-1 are the most widely used LVs. Genome Lentiviruses are enveloped viruses that have their RNA genome encapsulated. The three main genes coding for viral proteins are env, gag and pol. The env gene codes for the envelope protein that determines the specific target cell. The protein coded by gag gene is the core structural protein of the viruses and has several vital functions. The pol gene encodes for the integrase and reverse transcriptase proteins. The HIV-1 genome also contains regulatory genes, tat and rev, along with various accessory genes (vif, vpr, vpu, nef) that support replication. The 5’ and 3’ long terminal repeat (LTR) regions are required for integration of viral DNA into host genome. Advantages LVs have several characteristics that make them a favorable option for transduction in laboratory and for gene therapy 3. LVs can: deliver significant amount of viral DNA per cell, and since it gets integrated into the genome, it is constitutively expressed. transduce both dividing and non-diving cells, be used to target a wide range of cell types, deliver complex genetic elements, do not express any viral proteins after transduction, have low genotoxicity and Vector manipulation and production is not complicated. Disadvantages LVs could possibly integrate genetic material into essential regions of host genome leading to disruption of normal function. This is particularly a major concern with respect to gene therapy, and was the cause of failure of clinical trials with γ-retroviruses counterparts 4. Another safety concern with gene therapy, particularly with using HIV-1 derived LVs, is generation of replication-competent lentiviruses (RCLs). Origin, Development and Construction of LVs The use of viral vectors was first initiated in 1970s. In 1976, parts of Simian virus 40 along with lambda phage were used to transduce cultured monkey cells 5. It was in early 1980s that HIV-1 was discovered and in 1996, recombinant HIV-1 came into consideration for the construction of LVs 6. Since then, a lot of work has been put into designing LVs that are incapable of producing infectious virus in the host. For this reason only the critical viral structural and functional sequences are provided in LV, and the ones that are provided are divided into different plasmids, some of which get into the genome of plasmid are hence encoded by the LV (cis) while others are only provided as proteins for making functional viruses that can infect cells once to deliver the sequence (trans). The sequences in trans are not encoded by the LVs. Also, overlaps between viral sequences are avoided, if not kept minimal. There are in general at least three plasmid used: a packaging plasmid, this provides sequences coded by gag and pol in trans, i.e., all the structural and enzymatic sequences which will generate a functional viral particle. a transfer or expression plasmid that has the genetic sequence to be introduced in cis in the non-coding region of the viral sequence. This plasmid also contains the viral packaging signal along with LTR regions with the promoter activity of 3’ LTR deleted. an envelope plasmid encoding an envelope glycoprotein that determines the kinds of cells the LV can target. These three different plasmids are made into LVs by transiently transfecting them into HEK239 or 293T cells, which secrete LV 2-3 days post transfection. Over the years, further modifications have been made to the plasmids to improve the safety of LV and increase their efficiency in transduction. Initially, the packaging plasmid had gag and pol along with the all the regulatory and accessory elements. This plasmid was attenuated by removing accessory genes but not regulatory genes 8. Another major accomplishment was the development of self-inactivating LVs that had a deletion in the 3’ LTR region 9. This dramatically increased the safety of the LVs. Furthermore, the envelope plasmid was modified. The G protein of the Vesicular Stomatitis Virus (VSV-G) envelope gene replaced HIV-1 env, as it has a broader cell host range than the HIV-1 env protein. Applications The various advantages of LV have led to its use in various fields of biological research. In one study, LVs were used to introduce double stranded siRNA against the HIV-1 co-receptor CCR5 in human peripheral blood lymphocytes to inhibit HIV-1’s ability to infect these cells 10. LVs were designed using principles discussed above. Added on, the LVs expressed GFP to easily track the transduced cells. F12 is the LV used in this study to introduce the siRNA along with GFP into cells (Figure ). The lacZ-siRNA was used as a negative control along with a weak anti-CCR5-siRNA(809) and a potent anti-CCR5-siRNA(186) sequence (Figure ). The potent CCR5 si-RNA resulted in up to 10-fold dreacease in CCR5 expression in these cells (Figure ) and also provided substantial protection from the CCR5-tropic HIV-1 virus (Figure ) Some its uses include: Immunization (Breckpot, Emeagi et al. 2008), in vivo imaging (Roet, Eggers et al. 2012) Generation of trasnsgenic mice (Baup, Fraga et al. 2010), transgene overexpression (Lopez-Ornelas, Mejia-Castillo et al. 2011), persistent gene silencing (Wang, Hu et al. 2012), stem cell modification (Sanchez-Danes, Consiglio et al. 2012), lineage tracking and site-directed gene editing (Lombardo, Genovese et al. 2007), various applications in cancer cells (Petrigliano, Virk et al. 2009), gene therapy. References 1. Escors, D. and K. Breckpot, Lentiviral vectors in gene therapy: their current status and future potential. Arch Immunol Ther Exp (Warsz), 2010. 58(2): p. 107-19. 2. Cattoglio, C., et al., Hot spots of retroviral integration in human CD34+ hematopoietic cells. Blood, 2007. 110(6): p. 1770-8. 3. Bauer, G., et al., In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors. Mol Ther, 2008. 16(7): p. 1308-15. 4. Manilla, P., et al., Regulatory considerations for novel gene therapy products: a review of the process leading to the first clinical lentiviral vector. Hum Gene Ther, 2005. 16(1): p. 17-25. 5. Goff, S.P. and P. Berg, Construction of hybrid viruses containing SV40 and lambda phage DNA segments and their propagation in cultured monkey cells. Cell, 1976. 9(4 PT 2): p. 695-705. 6. Naldini, L., et al., In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector. Science, 1996. 272(5259): p. 263-7. 7. Zufferey, R., et al., Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nat Biotechnol, 1997. 15(9): p. 871-5. 8. Dull, T., et al., A third-generation lentivirus vector with a conditional packaging system. J Virol, 1998. 72(11): p. 8463-71. 9. Romano, G., et al., Human immunodeficiency virus type 1 (HIV-1) derived vectors: safety considerations and controversy over therapeutic applications. Eur J Dermatol, 2003. 13(5): p. 424-9. 10. Qin, X.F., et al., Inhibiting HIV-1 infection in human T cells by lentiviral-mediated delivery of small interfering RNA against CCR5. Proc Natl Acad Sci U S A, 2003. 100(1): p. 183-8.